Summary Analysis of gene expression in pathogens and investigation of host-pathogen interactions are a critical step in fueling new drug and vaccine discovery projects. In this study, we focus on the protozoan parasite Leishmania major, the causative agent of human cutaneous leishmaniasis. While significant progress has been made over the past 20 years toward understanding the molecular and cellular basis for intracellular survival of Leishmania, we still lack information regarding the biology of intracellular amastigotes. In particular, little is known about gene expression in the parasite prior to and following phago-lysosomal fusion and the genes required to facilitate adaptation to, and survival within, the host cell. Furthermore, genes that are uniquely activated and expressed in the human macrophage, in the context the infection, have not been systematically determined. The recent emergence of next-generation sequencing technologies opens up unprecedented opportunities to gain an in-depth view of the transcriptome of L. major and the macrophage in the context of infection. The goal of this study is to obtain the first comprehensive transcriptome profiling data for the three main developmental stages of L. major (Aim 1) and to simultaneously analyze gene expression programs in the intracellular amastigote stage of L. major along with the trancriptional response of the infected human host cell, the macrophage (Aim 2). These studies should provide insight into host defense mechanisms initiated by macrophages and lead to a better understanding of what the macrophage sees on the parasite as it is being ingested. To further examine the interactions between the parasite and the human macrophage, we will identify a subset of important Leishmania-human protein-protein interaction (PPI) partners using well-established yeast two-hybrid screens (Aim 3). Very few experimental studies have investigated host-pathogen PPIs. Extending those to the intracellular parasite Leishmania will not only allow the identification PPIs that enable this parasite to infect its host cells, acquire nutrients and evade its immune defenses, but will also provide a more global functional view of pathogenesis in general. In summary, this study will provide the first massive-scale and simultaneous interrogation of the transcriptomes of an intracellular protozoan parasite and its macrophage host cell and will provide a first look at the infectome, the part of a host cell's genome and proteome that is important for infection by a pathogen as well as the part of the pathogen's genome/proteome that allows it to subvert the functions of some host cell receptors, signaling proteins and molecular machinery. .